Proteins with an attached short peptide of acidic amino acids

ABSTRACT

Disclosed are a fusion protein comprising enzyme N-acetylgalactosamine-6-sulfate sulfatase and a short peptide consisting of 4-15 acidic amino acids attached to the enzyme on its N-terminal side, a pharmaceutical composition containing the fusion protein, and a method for treatment of type A Morquio disease using the fusion protein. Compared with the native enzyme protein, the fusion protein exhibits higher transferability to bone tissues and improved, higher stability in the blood.

TECHNICAL FIELD

The present invention relates to endowing a protein with in vivobone-tissue targeting ability and improvement of its stability in theblood. More specifically, the present invention relates to endowing aprotein with bone-tissue targeting ability and improvement of itsstability by means of addition of a short peptide consisting of acidicamino acids to the N-terminus of the protein.

BACKGROUND ART

It has been reported that acidic peptide chains consisting of asparticacid and/or glutamic acid molecules have high bonding affinities forhydroxyapatite, one of the component materials of the bone (1, 2).Making use of this property, techniques have been reported by whichthose acidic peptide chains are attached to steroid hormones (sexhormones or protein anabolic hormones, etc.), which are used for bonediseases such as osteoporosis, for endowing those steroid hormones withbone-tissue targeting ability (Japanese Patent Application PublicationNo. 2000-327583)(3). Further techniques have been reported by whichpeptide chains made of carboxylated amino acid derivatives having threeor more carboxyl groups per molecule were attached to and used asbone-targeting, drug-transporting carriers for steroid hormones,methotrexate, anti-cancer antibiotics, alkylating agents or cell growthfactors (Japanese Patent Application Publication No. 2002-3407)(4).

Meanwhile, there is a problem that pharmaceutical preparations ofphysiologically active proteins like enzymes and peptide hormones aregenerally made unstable when they are administered to the body, and thusundergo relatively rapid inactivation by, e.g., enzymatic degradation.For stabilizing pharmaceutical preparations of physiologically activeproteins in the body, a method is known which is based on coupling theproteins to polyethylene glycol (Japanese Patent No. 2852127)(5).

Among numbers of diseases caused by congenital anomaly, there is type IVA mucopolysaccharidosis (hereinafter referred to as type A Morquiodisease). In mucopolysaccharidosis, which forms a group of lysosomaldiseases caused by a deficiency of enzymes necessary for the metabolismof glycosaminoglycan (hereinafter referred to as GAG), accumulation ofGAG occurs in affected part of the tissue as a result of the deficiencyof that enzyme. Although its clinical symptoms vary among individualpatients, its common characteristic pathologies include swelling ofcells caused by the accumulation of GAG in lysosomes, hypertrophy oforgans, destruction of tissues and failing organs. Progressiveaccumulation of GAG is noted and clinical symptoms are, in majority ofcases, also chronic and progressive.

Type A Morquio disease is an autosomal recessive genetic disease causedby an anomaly in the gene for a lysosomal enzyme,N-acetylgalactosamine-6-sulfate sulfatase (hereinafter referred to asGALNS) and is classified as type IV A mucopolysaccharidosis. GALNS is anenzyme that hydrolyses the sulfate groups of chondroitin-6-sulfate andkeratan sulfate, which are species of GAG, and the deficiency of theenzyme causes intra-tissue deposition of GAG and its increased excretionin the urine. One of the clinical characteristics of Type A Morquiodisease is bone dysplasia, and thus short statute, scoliokyphasis,brevicollis, coxa valga, and articular hyperextension have been reportedto occur. Also reported are corneal opacity, deafness and cardiacvalvular disorders. On the one hand, one of its characteristics that isquite different from Hunter's syndrome and Hurler's syndrome is that nopsychomotor retardation is observed in patients with type A Morquiodisease (6).

No effective remedy is currently available for type A Morquio disease,and bone marrow transplantation provides no more than a marginalimprovement of osteopathy. Thus, most of its treatment is addressed tosymptomatic therapy or control of symptoms, like orthopaedic treatmentto prevent dislocation in upper cervical vertebrae. On the other hand,as main symptoms are localized in the bone and joints in type A Morquiodisease, it is expected that the quality of life of the patients couldbe greatly improved if their osteopathy is alleviated. Whilesubstitution therapy with enzyme GALNS is contemplated for type AMorquio disease, substitution therapy using native GALNS is not expectedto give any satisfactory effect considering its rapid inactivation inthe body and low rate of its transfer to bone tissues including growingcartilage.

DISCLOSURE OF INVENTION

Against the above-mentioned background, an objective of the presentinvention is to achieve increased rate of delivery, to bone tissues, ofphysiologically active proteins such as enzymes to be administered to apatient, through endowing them with a targeting ability to that tissue.

Another objective of the present invention is to increase in vivostability of physiologically active proteins administered to a patientsuch as enzymes. When an acidic short peptide was attached to theN-terminus of enzyme GALNS, the inventors found that it unexpectedlyimproved in great deal the in vivo stability of GALNS and allowednotable transfer of GALNS to bone tissues. The present invention wascompleted upon the findings.

Thus, the present invention provides:

1. A fusion protein comprising

-   -   a physiologically active protein and    -   a short peptide which consists of 4-15 acidic amino acids and is        attached to the physiologically active protein on the N-terminal        side thereof.

2. The fusion protein according to 1 above, wherein the physiologicallyactive protein is an enzyme protein.

3. The fusion protein according to 1 above, wherein the physiologicallyactive protein is N-acetylgalactosamine-6-sulfate sulfatase.

4. The fusion protein according to one of 1 to 3 above, wherein theshort peptide is attached to the N-terminus of the physiologicallyactive peptide via a linker peptide.

5. A method for enhancing the transferability of a physiologicallyactive protein from the blood to bone tissues, wherein the methodcomprises converting the physiologically active protein into a fusionprotein comprising the physiologically active protein and a shortpeptide which consists of 4-15 acidic amino acids and is attached to thephysiologically active protein on the N-terminal side thereof.

6. The method according to 5 above, wherein the physiologically activeprotein is an enzyme protein.

7. The method according to 5 above, wherein the physiologically activeprotein is N-acetylgalactosamine-6-sulfate sulfatase.

8. The method according to one of 5 to 7 above, wherein the shortpeptide is attached to the N-terminus of the physiologically activepeptide via a linker peptide.

9. A method for increasing the stability of a physiologically activeprotein in the blood, wherein the method comprises converting thephysiologically active protein into a fusion protein comprising

-   -   the physiologically active protein and    -   a short peptide which consists of 4-15 acidic amino acids and is        attached to the physiologically active protein on the N-terminal        side thereof.

10. The method of according to 9 above, wherein the physiologicallyactive protein is an enzyme.

11. The method according to 9 above, wherein the physiologically activeprotein is N-acetylgalactosamine-6-sulfate sulfatase.

12. The method according to one of 9 to 11 above, wherein the shortpeptide is attached to the N-terminus of the physiologically activepeptide via a linker peptide.

13. A pharmaceutical composition comprising a fusion protein comprising

-   -   N-acetylgalactosamine-6-sulfate sulfatase and    -   a short peptide which consists of 4-15 acidic amino acids and is        attached to N-acetylgalactosamine-6-sulfate sulfatase on the        N-terminal side thereof.

14. The pharmaceutical composition according to 13 above, wherein theshort peptide is attached to the N-terminus of the physiologicallyactive protein via a linker peptide.

15. A method for treatment of type A Morquio disease, comprisingadministering to a human patient therewith a therapeutically effectiveamount of a fusion protein comprising

-   -   N-acetylgalactosamine-6-sulfate sulfatase and    -   a short peptide which consists of 4-15 acidic amino acids and is        attached to N-acetylgalactosamine-6-sulfate sulfatase on the        N-terminal side thereof.

16. The method for treatment according to 15 above, wherein the shortpeptide is attached to the N-terminus of N-acetylgalactosamine-6-sulfatesulfatase via a linker peptide.

Comparing with native physiologically active proteins, the presentinvention described above provides physiologically active fusionproteins with increased stability in the blood when administered to apatient such as a human patient as well as enhanced targeting ability tobone tissues, and, in particular, a GALNS fusion protein modified insuch a manner, and further provides a pharmaceutical composition usefulfor the treatment of type A Morquio disease, as well as a method for thetreatment of type A Morquio disease.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 is a schematic diagram illustrating pCXN vector and the cloningsite in the vector for the cDNA encoding native GALNS or the GALNSfusion protein.

FIG. 2 illustrates a first half group of the steps for the constructionof an expression vector pCXN-p97-NBT-GALNS used for the production of aGALNS fusion protein.

FIG. 3 illustrates a second half group of the steps for the constructionof an expression vector pCXN-p97-NBT-GALNS used for the production ofthe GALNS fusion protein.

FIG. 4 shows the result of SDS-PAGE analysis of purified native GALNSand the GALNS fusion protein.

FIG. 5 shows the images of the stained GALNS fusion protein, incomparison with native GALNS, transferred to the myeloid tissue.

FIG. 6 is a graph showing the time profiles of the blood activity levelsof native GALNS and the GALNS fusion protein after they areintravascularly administered in an equivalent amount.

BEST MODE FOR CARRYING OUT THE INVENTION

The term “acidic amino acid” referred to in the present invention meansglutamic acid or aspartic acid. As the employment of these acidic aminoacids in the present invention are for the purpose of constructing anacidic short peptide, they may be used in any arbitrary combinationincluding a simple use of one or the other of them alone for theconstruction of such a short peptide. The number of the acidic aminoacids forming a short peptide is preferably 4-15, more preferably 4-12,and still more preferably 4-8.

A short peptide consisting of acidic amino acids may be directlyattached to the N-terminus of a physiologically active protein via apeptide bond or the like, or, instead, it may be attached via a linkerpeptide.

In the present invention, “a linker peptide” is not an indispensablecomponent, for it is usable only for convenience in attaching a shortpeptide consisting of acidic amino acids to the N-terminus of aphysiologically active protein. Where it is used, a linker peptide maybe a short peptide consisting, e.g., preferably of 15 or less, morepreferably of 10 or less, and still more preferably of 6 or less aminoacids. Such a linker that consists of a single amino acid molecule andlinking between the acidic short peptide and the physiologically activeprotein via peptide bonds is also included in the definition of a linkerpeptide. A linker peptide may be made of any one or more amino acids asdesired.

In the present invention, though there is no specific limitation as tothe method for attaching an acidic short peptide and a physiologicallyactive protein, it is of advantage, e.g., to form and use a transformantcell expressing the fusion protein consisting of the short peptide andthe physiologically active protein.

A fusion protein of the present invention may include a non-acidic aminoacid or a sequence of several (e.g., 3) non-acidic amino acids at theN-terminus of the short peptide consisting of acidic amino acids.

A fusion protein of the present invention, in particular a fusionprotein comprising enzyme N-acetylgalactosamine-6-sulfate sulfatase anda short peptide, may be prepared in the form of a pharmaceuticalcomposition containing the fusion protein dissolved or dispersed in apharmaceutically acceptable carrier well known to those who are skilledin the art, for parenteral administration by, e.g., intravenous,subcutaneous or intramuscular injection or by intravenous drip infusion.

For a pharmaceutical composition for parenteral administration, anyconventional additives may be used such as excipients, binders,disintegrants, dispersing agents, lubricants, diluents, absorptionenhancers, buffering agents, surfactants, solubilizing agents,preservatives, emulsifiers, isotonizers, stabilizers, solubilizers forinjection, pH adjusting agents, etc.

A fusion protein of the present invention, in particular a fusionprotein comprising enzyme N-acetylgalactosamine-6-sulfate sulfatase anda short peptide, may be used advantageously in place of the conventionalnative enzyme in a substitution therapy for the treatment of type AMorquio disease. In the treatment, the fusion protein may beadministered intravenously, subcutaneously or intramuscularly. Doses andfrequencies of administration is to be determined by the physician incharge in accordance with the condition of his or her patient.

EXAMPLES Method for Construction of Expression Vector

Vector pCXN had been constructed in accordance with a literature (7) andwas offered to us by Prof. Miyazaki at Osaka University. An expressionvector for native human GALNS, pCXN-GALNS, was one that had beenreported by Tomatsu et al. in 1991 (8). An expression vector for humanGALNS to the N-terminus of which is attached (via a linker peptide) ashort peptide (N-terminal bone tag: NBT) consisting of acidic aminoacids (NBT-GALNS) was constructed starting with pCXN-GALNS in thefollowing manner. FIGS. 2 and 3 schematically illustrate the process forconstruction.

By cleaving pCXN-GALNS with EcoRI, human GALNS cDNA was recovered. Usingthis as a template, PCR was carried out using IA-Taq (Takara) to amplifyΔsig GALNS cDNA (nt 79-1569 in SEQ ID NO:1, which is the sequence leftafter removal of nt 1-78 corresponding to the secretion signal from theORF region.) to the 5′-terminus of which is attached an AgeI cleavagesequence. The PCR was carried out according to the instruction for useof LA-Taq, i.e., through the cycles consisting of 30 seconds at 94° C.,(30 seconds at 94° C., 30 seconds at 60° C., and 2 minutes at 72°C.)×25, and then 3 minutes at 72° C., with primer 1 (SEQ ID NO:2) andprimer 2 (SEQ ID NO:3). The cDNA thus amplified was inserted into thepT7Blue vector (Novagen) to construct pT7-Δsig GALNS.

An N-terminal bone tag (NBT) cDNA to be attached to the 5′-terminus thenwas constructed by PCR using LA-Taq (Takara). Briefly, employing primer3 (SEQ ID NO:4) and primer 4 (SEQ ID NO:5), which contained portionsthat were complementary to each other, PCR was performed through thecycles of 30 seconds at 94° C., (30 seconds at 94° C., 30 seconds at 60°C., and 30 seconds at 72° C.)×20, and then one minute at 72° C. The cDNAthus amplified was cloned into the pT7Blue vector to construct pT7-NBT.

A human GALNS cDNA recovered as a fragment of pT7-Δsig GALNS cleavedwith AgeI and XbaI was inserted into the AgeI-XbaI site of pT7-NBT toconstruct pT7-NBT-GALNS. Then, pT7-NBT-GALNS was cleaved with BclI,blunt-ended with T4 DNA polymerase by addition of nucleotides to itssticky ends, and cleaved with XbaI to recover the NBT-GALNS cDNA.

pST-RAP-GUSB (A vector containing the p97 signal sequence. This wasprovided by Tomatsu at Saint Louis University) was cleaved with BamHIand XbaI, into which then was inserted the NBT-GALNS cDNA recoveredabove to construct pST-p97-NBT-GALNS.

pST-p97-NBT-GALNS was cleaved with EcoRI to recover the p97-NBT-GALNScDNA, which then was inserted into the EcoRI site of pCXN to constructan NBT-GALNS expression vector, pCXN-p97-NBT-GALNS. The DNA sequence ofthe region containing the NBT-GALNS cDNA of the expression vector andthe corresponding amino acid sequence are shown as SEQ ID NOs:6 and 7,respectively.

SEQ ID NO:6 is part of the sequence containing the NBT-GALNS cDNA ofpCXN-p97-NBT-GALNS. Nt 13-19 encode the p97 signal sequence, nt 73-90 apoly Glu, nt 91-108 a linker sequence, and nt 109-1596 GALNS without thesignal sequence.

SEQ ID NO:7 is the NBT-GALNS with the p97 signal sequence. Aa 1-19: p97signal sequence, aa 21-26: poly Glu, aa 27-32: linker sequence, aa33-528: GALNS without signal sequence.

The protein set forth as SEQ ID NO:7 contains the p97 secretion signalsequence. The signal sequence is removed during secretion process fromthe cell and a fusion protein consisting of aa 20-528 is thus recoveredas NBT-GALNS in the medium.

P97 is a cell-surface glycoprotein occurring most human melanomas andits signal sequence consists of 19 amino acids (9). The aforementionedpCXN-p97-NBT-GALNS containing the cDNA encoding this signal sequence mayalso be constructed by the following method. Briefly, a cDNA containingthe p97 signal sequence is synthesized through the process of primersannealing and PCR amplification. LA-Taq is used as an enzyme for PCR. Asprimers having mutually complementary portions, primer 4 (SEQ ID NO:8)and primer 5 (SEQ ID NO:9) are used. PCR is performed through the cyclesof 30 seconds at 94° C., (30 seconds at 94° C., 30 seconds at 60° C., 30seconds at 72° C.)×20, and one minute at 72° C. The amplified cDNAcontaining the p97 signal sequence is cleaved with BamHI and EcoRI. ThepCXN vector is cleaved with EcoRI, into which are simultaneouslyincorporated the aforementioned NBT-GALNS cDNA recovered after theenzyme treatment and the cDNA for the p97 signal sequence to constructpCXN-p97-NBT-GALNS.

SEQ ID NO:8 is a forward primer 5, in which nt 1-5 comprise a randomsynthetic sequence, and nt 6-52 comprise part of the sequence encodingp97 signal.

SEQ ID NO:9 is a reverse primer, in which nt 1-6 comprise a randomsequence, and nt 7-51 comprise part of the sequence encoding p97 signal.

[Establishment of Expression Cell]

Nunclon ΔMultiDish 6 Well was inoculated with CHO-K1 cells. After anovernight culture in DMEM/F12/FBS medium, either pCXN-p97-NBT-GALNS orpCXN-GALNS was introduced into the cells using the Lipofectamine 2000reagent. For experimental procedures, the instruction manual attached tothe Lipofectamine 2000 reagent was followed. After a two-day incubationat 37° C. in 5% CO₂, the cells were added to 75-cm² tissue cultureflasks (Iwaki) and incubated until colonies of resistant cells wereformed with Geneticin (Gibco) added to the DMEM/F12/FBS medium at thefinal concentration of 1 mg/mL. After formation of colonies wasconfirmed under a microscope, cells which exhibited stable expressionwere cloned by the limiting dilution-culture method. Screening forexpression cells were performed by sulfatase assay of the culturesupernatants. The NBT-GALNS expression cell line was designatedCHO-NBT-GALNS40, and the human native GALNS expression cell lineCHO-GALNS14. The established cell lines were subcultured in the DMEM/F12medium (Gibco) supplemented with 10% fetal bovine serum (Thermo Trace)and 0.2 mg/mL Geneticin.

[Culture of Cells Expressing NBT-GALNS and GALNS]

CHO-GALNS 14 cells (cells expressing human native GALNS) andCHO-NBT-GALNS 40 cells (cells expressing NBT-attached human GALNS) werecultured as follows. Briefly, a DMEM/F12 medium (Gibco) supplementedwith 10% fetal bovine serum (Thermo Trace) and 0.2 mg/mL geneticin wasused as the subcultivation medium (DMEM/F12/FBS/G), and a EX-CELL 325 PFmedium containing 2 mM glutamine, 10 mg/L hypoxantine and 4 mg/Lthymidine was used as the production medium (EX-CELL 325 G/H/T). All thecultivation was performed at 37° C. in 5% CO₂. Cells in DMEM/F12/FBSwere added to 18 of 225 cm² tissue culture flasks (Iwaki) and culturedovernight so that 80-100% confluency would be reached the following day.The culture supernatant was removed and the cells were washed twice withPBS(−). After the medium was replaced with about 50 mL/flask of EX-CELL325 G/H/T, culture was continued for three days. The culture supernatantwas recovered and immediately frozen at −20° C. and stored. All thecells were detached with 0.25% Trypsin-EDTA (Gibco), suspended in 5 L ofDMEM/F12/FBS, added evenly to 48 of Nunclon ΔTriple Flasks (Nunc) andcultured overnight. The culture supernatant was removed, and the cellswashed twice with PBS(−). After the medium was replaced with about 100mL/flask of EX-CELL 325 G/H/T, culture was continued for 3 days. Theculture supernatant was recovered and immediately frozen and stored at−20° C. Following the above procedures, 6 L of culture supernatant wasrecovered per operation.

[Purification of Native GALNS]

The aforementioned culture supernatant was thawed in a water bath, andfiltered through a 0.22 μm polyethersulfone membrane (1000 mL FilterSystem, Corning Inc.) to remove insoluble contaminants in the culturesupernatant. The filtered culture supernatant was concentrated 20-foldby ultrafiltration using regenerated cellulose membrane(Prep/Scale-TFF-1 30 kDa MWCO, MILLIPORE Corporation). In preparationfor ion-exchange chromatography, the concentrated culture supernatantwas then dialyzed against 20 mM Tris-HCl, 50 mM NaCl, pH 7.8.

The buffer-exchanged and concentrated culture supernatant was loadedonto a HiPrep DEAE 16/10 DEAE FF column (Amersham Biosciences)(bedvolume: 20 mL) that had been equilibrated with 20 mM Tris-HCl, 50 mMNaCl, pH 7.8. After washing the column with the equilibration buffer,fractions that were eluted with 20 mM Tris-HCl, 150 mM NaCl, pH 7.8(flow rate: 5 mL/min) were collected.

In preparation for Q Sepharose chromatography, the collected fractionswere dialyzed against a dialysate of 10 mM MES, 50 mM NaCl, pH 5.5. Thefractions collected by DEAE Sepharose chromatography andbuffer-exchanged were loaded onto a HiPrep Q 16/10 Q FF column (AmershamBiosciences)(bed volume: 20 mL) that had been equilibrated with 10 mMES, 50 mM NaCl, pH 5.5 and passing through fractions were collected(flow rate: 5 mL/min). The column was washed with the equilibrationbuffer and washing fractions were also collected (flow rate: 5 mL/min).

In preparation for Sephacryl S-200 chromatography, the collectedfractions were concentrated by ultrafiltration using regeneratedcellulose membrane (Amicon Ultra-15, 30 kDa MWCO). The concentratedfractions collected by the Q Sepharose chromatography were loaded onto aHiPrep 16/60 Sephacryl S-200 HR column (Amersham Biosciences) that hadbeen equilibrated with 10 mM MES, 150 mM NaCl, pH 5.5 and fractionscorresponding to the aimed peak were collected.

SDS-PAGE gel electrophoresis of the NBT-GALNS collected through thesteps described above confirmed that it had been purified enough toappear as a single band (FIG. 4).

At each step of the purification, fractions containing native GALNS weredetected by sulfate assay described later.

[Purification of NBT-GALNS]

As NBT-GALNS was expected to differ from native GALNS in physicochemicalproperties and have a high affinity for hydroxyapatite, it was purifiedby a method described below which included hydroxyapatitechromatography.

The aforementioned culture supernatant was thawed in a water bath, andfiltered through a 0.22 μm polyethersulfone membrane (1000 mL FilterSystem, Corning Inc.) to remove insoluble contaminants in the culturesupernatant. The filtered culture supernatant was concentrated 20-foldby ultrafiltration using regenerated cellulose membrane(Prep/Scale-TFF-1 30 kDa MWCO, MILLIPORE Corporation). In preparationfor ion-exchange chromatography, the concentrated culture supernatantwas then dialyzed against a dialysate of 20 mM Tris-HCl, 50 mM NaCl, pH7.8.

The buffer-exchanged and concentrated culture supernatant was loadedonto a HiPrep DEAE 16/10 DEAE FF column (Amersham Biosciences)(bedvolume: 20 mL) that had been equilibrated with 20 mM Tris-HCl, 50 mMNaCl, pH 7.8. After washing the column with the equilibration buffer,fractions that were eluted with 20 mM Tris-HCl, 150 mM NaCl, pH 7.8(flow rate: 5 mL/min) were collected. In preparation for Q Sepharosechromatography, the collected fractions were dialyzed against 10 mM MES,50 mM NaCl, pH 5.5 for buffer exchange.

The fractions collected by DEAE Sepharose chromatography andbuffer-exchanged were loaded onto a HiPrep Q 16/10 Q FF column (AmershamBiosciences)(bed volume: 20 mL) that had been equilibrated with 10 mMES, 50 mM NaCl, pH 5.5. After washing the column with the equilibrationbuffer, fractions eluted with 10 mM MES, 250 mM NaCl, pH 5.5 werecollected (flow rate: 5 mL/min).

Fractions collected by Q Sepharose chromatography were loaded onto ahydroxyapatite column, Econo-Pac CHT-II cartridge column (Bio-Rad)(bedvolume: 5 mL) that had bee equilibrated with 1 mM phosphate buffer, 10mM MES, 50 mM NaCl, pH 5.5. After washing the column with theequilibration buffer (flow rate: 0.5 mL/min), fractions eluted with 500mM phosphate buffer, 10 mM MES, 50 mM NaCl, pH 5.5 was collected (flowrate: 0.5 mL/min). In preparation for Sephacryl S-200 chromatography,the collected fractions were concentrated by ultrafiltration usingregenerated cellulose membrane (Amicon Ultra-15 30 kDa, MWCO).

The concentrated fractions obtained by CHT-II hydroxyapatitechromatography were loaded onto a HiPrep 16/60 Sephacryl S-200 HR column(Amersham Biosciences) that had been equilibrated with 10 mM MES, 150 mMNaCl, pH 5.5 and fractions corresponding to the aimed peak werecollected.

SDS-PAGE gel electrophoresis of the NBT-GALNS collected through thesteps described above confirmed that it had been purified enough toappear as a single band (FIG. 4).

At each step of the purification, fractions containing GALNS weredetected by sulfate assay described below.

[Sulfate Assay]

To 10 μL of a sample was added 100 μL of a substrate solution [5 mM4-methylumbelliferyl sulfate (Sigma), 5 mM sodium acetate (Wako PureChemical Industries), 0.05% BSA (Sigma), pH 4.4]. After one-hourreaction at 37° C., 190 μL of a stop solution [332 mM glycine (Wako PureChemical Industries), 208 mM sodium carbonate (Wako Pure ChemicalIndustries), pH 10.7] was added and fluorescence was measured on afluorescence plate reader (Molecular Device) at the wavelengths of 460nm (em)/355 nm (ex). As a control, 4-methylumbelliferone (Sigma) wasused which was diluted with a solution prepared by mixing the substratesolution and the stop solution in the proportion described above.Starting with 1 mM, 7 steps of twofold dilution were made.

[SDS Polyacrylamide Gel Electrophoresis]

1) SDS Polyacrylamide Gel Electrophoresis and Staining

Samples were treated with SDS using Laemmli Sample Buffer (Bio-Rad) inthe presence of 5% β-mercaptoethanol (Bio-Rad) and subjected to SDS-PAGEin PAG mini “DAIICHI” 12.5 (Daiichi Pure Chemicals). For staining, SYPRORUBY GEL STAIN (Biorad) or 2D-silver stain reagent-II “DAIICHI” (DaiichiPure Chemicals) was used.

[Preparation of Antibodies]

Polyclonal and monoclonal antibodies were prepared by a conventionalmethod through immunization of mice with native GALNS as the antigen.

[Immunostaining of Bone Tissues]

GALNS or NBT-GALNS, both purified, was administered to four-month oldmale C57BL mice in the tail vein at a dose of 250 U/g body weight. Twohours after administration, tissue specimen slides were prepared forbone slices of the mice including femur, knee joint, and cervicalvertebra. Briefly, the slide were immersed in a xylene bath for 3minutes, taken out and then transferred to another xylene bath. Thisstep was repeated 5 times. The xylene-treated slides were immersed in99.5% ethanol for 3 minutes, taken out and then transferred to anotherethanol bath. This step was repeated 3 times. The slides were washed 3times in the same manner with 95% ethanol and immersed in a phosphatebuffer for 15 minutes. The phosphate buffer was wiped out of the slides.The slices then were covered with methanol containing 3% hydrogenperoxide and allowed to stand for 5 minutes at room temperature toinactivate intrinsic peroxidases. The slides then were washed withphosphate buffer. The slices on the slides were covered with theblocking reagent A included in Histofine immunohistochemical stainingreagents (Nichirei) and allowed to stand for 60 minutes at roomtemperature. After this treatment, the slides were washed with phosphatebuffer.

The slices were covered with an anti-GALNS monoclonal antibody solutionand allowed to stand for 15 hours at 4° C. After the reaction, theslides were washed with phosphate buffer. The slices on the slides werecovered with the blocking reagent B included in Histofineimmunohistochemical staining reagents (Nichirei) and allowed to standfor 10 minutes at room temperature. After the treatment, the slides werewashed with phosphate buffer. The slices on the slides were covered withSimpleStain Mouse MAX-PO (Mouse) (Nichirei), which was aperoxidase-conjugated secondary antibody against mouse primaryantibodies, and allowed to stand for 5 minutes at room temperature.After the reaction, the slides were washed with phosphate buffer. Theslices on the slides were covered with SimpleStain DAB solution(Nichirei) and allowed to develop color while monitoring the strength ofthe color developed under a microscope. The reaction ofcolor-development was stopped by washing with purified water. The resultof staining is shown in FIG. 5. While signals of intense stained spotswere observed in the myeloid tissue from the mice administered withNBT-GALNS, hardly any signals were observed in the myeloid tissues fromthe mice administered with native GALNS. The results demonstrate thatNBT-GALNS, compared to native GALNS, has remarkable highertransferability to the bone tissue when administered to the circulatingblood.

[Method for Measurement of GALNS-Specific Enzyme Activity]

Measurement of GALNS activity in the blood after intravenousadministration of native- or NBT-GALNS to mice was performed as follows.Briefly, a 10-μL plasma sample was added to 18 μL of a solution of 10 mM4-Methylumbelliferyl-beta-D-Galactopyranoside-6-Sulfate (4MUF-Gal-6-S)(Toronto Research Chemicals Inc.) which had been prepared using adetermination buffer (100 mM NaCl, 100 mM CH₃COONa, pH 4.3), andreaction was allowed for 6 hours at 37° C. Then, 2 μL of 10 mg/mLBeta-Galactosidase solution (SIGMA) which had been prepared using thedetermination buffer were added, and allowed to react for 1 hour at 37°C. 970 μL of a stop buffer (1 M Glycine-HCl, pH 10.5) then were addedand mixed to stop the enzyme reaction. A 200-μL aliquot of the enzymereaction was transferred to a 96-well plate, and excitation 355nm/emission 460 nm was measured on a fluorescence plate reader(Molecular Device) fmax.

[Stability in the Blood]

Per 1 g of body weight, 250 U of GALNS or NBT-GALNS, both purified, wereadministered to 3 male, 4-month old C57BL mice per group in the tailvein. Samples of venous blood were collected at 2, 5, 10, 20, 30, 60 and120 minutes after the administration, and GALNS activity in the serumwas measured. The results are shown in FIG. 6. Comparison betweenNBT-GLNS-administered group and the native GALNS-administered grouprevealed that the enzyme activity in the blood at 2 minutes after theadministration was 2-fold higher in the NBT-GALNS-administered groupthan the native GALNS-administered group. And, while the enzyme activityin the blood at 20 minutes after the administration was almostdisappeared in the native GALNS-administered group, theNBT-GALNS-administered group retained activity levels, which were evenhigher than the activity levels found in the native GALNS-administeredgroup at 2 minutes after the administration, and showed much slowerreduction of activity afterwards than that found in the nativeGALNS-administered group. The results demonstrate that the stability ofGALNS in the body is remarkably increased by attaching a short peptideof acidic amino acid to native GALNS. To the best of the inventors'knowledge, there was no report indicating that acidic short peptideadded to GALNS or other physiologically active proteins improved thestability of such proteins in the body. This finding provides novelmeans for stabilization of different therapeutically useful enzymes,peptide hormones and other physiologically active proteins in the bodyafter they are administered in the blood.

INDUSTRIAL APPLICABILITY

The present invention enables production of physiologically activeproteins which have improved transferability to the bone tissues andincreased stability in the body. The present invention also providesmethod and pharmaceutical composition for treatment of type A Morquiodisease.

REFERENCES

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The invention claimed is:
 1. A fusion protein consisting of SEQ ID NO:7.
 2. A pharmaceutical composition comprising a pharmaceuticallyacceptable carrier and a fusion protein consisting of SEQ ID NO:
 7. 3.The fusion protein according to claim 1, which has increased stabilityin the blood and enhanced transferability from the blood to bone tissuescompared with native N-acetylgalactosamine-6-sulfate sulfatase.
 4. Thepharmaceutical composition according to claim 2, wherein said fusionprotein has increased stability in the blood and enhancedtransferability from the blood to bone tissues compared with nativeN-acetylgalactosamine-6-sulfate sulfatase.
 5. A fusion proteinconsisting of amino acids 20-528 of SEQ ID NO:
 7. 6. The fusion proteinaccording to claim 1, which has been purified.
 7. The fusion proteinaccording to claim 1, which has enhanced in vivo stability compared tonative GALNS.